PLEK2 Promotes The Occurrence And Metastasis Of Osteosarcoma By Activating The PI3K/AKT Signaling Pathway

Osteosarcoma(OS)is a malignant cancer that often occurs in adolescents.The etiology of osteosarcoma is multifaceted,but little is known about it.Research into the molecular mechanisms of this disease will lead to targeted cancer therapy.Pleckstrin-2(PLEK2)is widely expressed in various tissues,and its overexpression helps to promote the proliferation of cancer cells,but the role of PLEK2 in OS has not been studied.Therefore,this paper focuses on the effect of PLEK2 expression on OS growth and metastasis and its potential mechanism.Objective:More and more evidences have confirmed that PLEK2 is an oncogene in many malignant tumors.The effect of PLEK2 expression on OS growth and metastasis was studied in 143 B osteosarcoma cells with PLEK2 knockdown and U-2OS osteosarcoma cells with PLEK2 overexpressed,and the potential mechanism was revealed.Methods:1.The expression of PLEK2 in OS tissues was analyzed by bioinformatics.2.PLEK2 expression in 4 OS cells and osteoblasts(h FBO1.19)was detected by Western blot and immunohistochemical analysis.3.The PLEK2 cell line was stably knocked down by 143 B,U-2OS stably overexpressed cell line was constructed,and the success of cell line construction was detected by Western blot.4.CCK-8 assay,colony formation assay and Ed U assay were used to detect whether the proliferation rate of knockdown/overexpressed PLEK2 cell line changed.5.Transwell assay and Wound healing assay were used to detect changes in the migration ability of knockdown/overexpressed PLEK2 cell lines.6.Western blot assay was used to detect the changes of ETM-related proteins(E-cadherin,N-cadherin and Vimentin)in PLEK2 cell lines.7.Single gene GSEA analysis was conducted to find the related signaling pathways of PLEK2 activation.8.Western blot assay was used to detect the changes of proteins in the PI3K/AKT signaling pathway of the knocked down/overexpressed PLEK2 cell line,as well as the effects of the agonist IGF-1 and inhibitor LY294002 on the knocked down/overexpressed PLEK2 cell line.9.In vivo nodule experiments were performed to verify whether the nodule size of PLEK2 cell lines was altered.Immunohistochemistry was used to analyze Ki6 expression in nodules after nodule formation.Result:1.PLEK2 expression is upregulated in OS cells and tissues.2.In vitro,PLEK2 promotes osteosarcoma(OS)proliferation.CCK-8 experiment and colony formation experiment showed that PLEK2 knockdown significantly inhibited the proliferation of OS cells,while PLEK2 overexpression promoted the growth of OS cells.The results of Ed U test showed that PLEK2 knockdown reduced the proportion of mitotic cells and vice versa.3.In vitro,PLEK2 promotes osteosarcoma(OS)metastasis by activating EMT.Transwell experiment showed that PLEK2 knockdown inhibited the migration and invasion of 143 B cells,while PLEK2 overexpression increased the migration and invasion of U-2 OS cells.Wound healing assay showed that PLEK2 could be suppressed by knocking down its migration distance,while in U-2 OS cells with overexpression of PLEK2,the opposite result was found.Western blot results showed that PLEK2 knockdown induced em T-related protein changes(e-cadherin,N-cadherin and Vimentin),suggesting its role in EMT inhibition in osteosarcoma cells.,while overexpressing PLEK2 can reverse this effect.4.PLEK2 is involved in the PI3K/AKT/m TOR signaling pathway.Single gene GSEA analysis showed that overexpression of PLEK2 could activate the PI3K/AKT/m TOR signaling pathway.Knocking down PLEK2 can reduce the phosphorylation level of PI3 K and AKT,and inhibit the PI3K/AKT/m TOR pathway and EMT process.IGF-1,an agonist added to the PI3K/AKT pathway,can reverse these effects.Overexpression of PLEK2 activates the PI3K/AKT/m TOR pathway and the EMT process,while LY294002,an inhibitor of the PI3K/AKT pathway,can reverse these effects.5.In vivo,PLEK2 gene down-regulation inhibits osteosarcoma(OS)growth.In vivo nodule experiment showed that PLEK2 knockdown significantly reduced tumor volume and inhibited OS growth.Immunohistochemical experiments further proved that PLEK2 could significantly down-regulate the expression of Ki67.Conclusion:This study explored the role of PLEK2 in osteosarcoma(OS)and revealed its underlying mechanism in vitro.It was found that the expression of PLEK2 was up-regulated in OS,and the increased expression of PLEK2 in OS cells could activate the PI3K/AKT/m TOR pathway,thereby promoting the occurrence and metastasis of tumors.The discovery of PLEK2 may provide a potential therapeutic target for OS.